Fabrication of impedimetric sensor based on metallic nanoparticle for the determination of mesna anticancer drug

Electrochemical impedance spectroscopy (EIS) is a highly effective technique for studying the surface of electrodes in great detail. EIS-based electrochemical sensors have been widely reported, which measure the charge transfer resistance (Rct) of redox probes on electrode surfaces to monitor the binding of target molecules. One of the protective drugs against hemorrhagic cystitis caused by oxazaphosphorine chemotherapy drugs such as ifosfamide, cyclophosphamide and trophosphamide is Mesna (sodium salt of 2-mercaptoethanesulfonate). The increase in the use of Mesna due to the high consumption of anti-cancer drugs, the determination of this drug in biological samples is of particular importance. So far, no electrochemical method has been reported to measure Mesna. In this research, a novel impedimetric sensor based on a glassy carbon electrode (GCE) modified with oxidized multiwalled carbon nanotubes (MWCNTs)/gold nanoparticle (AuNPs) (denoted as Au NPs/MWCNTs/GCE) for impedimetric determination of Mesna anticancer drug was developed. The modified electrode materials were characterized by field emission scanning electron microscopy (FESEM), energy dispersive X-ray (EDX), and EIS. The electrochemical behavior of Mesna at the surface of Au NPs/MWCNTs/GCE was studied by an impedimetric method. The detection mechanism of Mesna using the proposed impedimetric sensor relied on the increase in the Rct value of [Fe (CN)6]3−/4− as an electrochemical probe in the presence of Mesna compared to the absence of Mesna as the analyte. Under the optimum condition, which covered two linear dynamic ranges from 0.06 nmol L−1 to 1.0 nmol L−1 and 1.0 nmol L−1 to 130.0 µmol L−1, respectively. The detection limit was 0.02 nmol L−1. Finally, the performance of the proposed sensor was investigated for Mesna electrochemical detection in biological samples.

Effective management of cancer requires optimizing the dose of anticancer drugs 1 . Mesna, the sodium salt of 2-mercaptoethanesulfonate, is commonly used to protect against hemorrhagic cystitis caused by oxazaphosphorine chemotherapy drugs like ifosfamide, cyclophosphamide, and trofosfamide. Mesna's free thiol group covalently binds to secondary metabolites of ifosfamide, preventing the formation of urotoxic acrolein. Acrolein binds to the bladder and kidney cells and causes cell death. Accurate determination of Mesna in biological samples is crucial due to its increasing use in cancer treatment and to minimize the side effects of the drug [2][3][4] . Therefore, it is essential to develop sensitive methods to measure Mesna levels in biological samples. Several methods, such as High-Performance Liquid Chromatography (HPLC) [5][6][7] , Raman scattering 8 , Spectrophotometry 9,10 , Fluorimetry 3,11 , and Argentometry 12 , have been proposed to estimate Mesna drug levels. However, these methods have some drawbacks, including low selectivity, long sample pretreatment, and extended analysis time. Electrochemical impedance spectroscopy (EIS) is a powerful technique widely used in analytical chemistry for designing electrochemical sensors. EIS is a label-free technique that can be used to study changes in the electrode surface, making it an ideal method for designing impedimetric sensors 13 . However, the electrode surface degrades after being used in the sample and needs to be modified before each determination. 14 . Until now, no electrochemical methods have been reported for determining Mesna levels, as per previous reports. Developing an electrochemical method for Mesna detection is necessary to overcome the limitations of the existing methods. In this technique, a sinusoidal potential wave is applied to a three-electrode system, and EIS data such as the real part of the impedance (Z real) , the imaginary part of the impedance (Z imaginary ), and phase shift (ϕ) at different frequencies are recorded. The obtained Nyquist plot, which plots Z imaginary against Z real , can be used to describe   www.nature.com/scientificreports/ where R is the ideal-gas constant, T treats the temperature in Kelvin, F is Faraday's constant, n is the number of electron transfers, A is the surface-area electrode (hereon: 0.0314 cm 2 ), and C * is the concentration of [Fe(CN) 6 ] 3−/4− (5 mmol L −1 ). Table 1 illustrated the more detailed data on EIS analysis obtained from spectra of Fig. 3 related to step-by-step electrode modification. According to Table 1, The k app value for the AuNPs/MWCNTs/GCE was calculated as 2.01 × 10 -3 cm s −1 , which was found to be 11.8 times higher than that of the bare electrode (1.7 × 10 -3 cm s −1 ). This indicates that electron transfer kinetics is significantly increased at the AuNPs/MWCNTs/GCE compared to the bare electrode. Additionally, the C dl value, which is a measure of the charge on the surface of the AuNPs/MWCNT/GCE, was calculated as 8.3 μF, which is 2.46 and 5.38 times higher than that at the MWCNTs/GCE (3.37 μF) and AuNPs/ GCE (1.54 μF) surfaces, respectively. The increase in the double-layer capacitance is attributed to the higher surface area due to the additional layer of modified electrode 30 . These results demonstrate that the proposed electrode exhibits an increase in electrochemical activity due to the increase in surface charge.
Surface area study. The effective surface area of the bare GCE, MWCNTs/GCE, Au NPs/ GCE, and Au NPs/MWCNTs/GCE were obtained by cyclic voltammetry for 0.5 mol L −1 KCl electrolyte solution containing 5 mmol L −1 [Fe(CN) 6 ] 3−/4− as a redox probe at different scan rates in the range of 5-300 mV. Randles-Sevcik Equation (Eq. 2) was used to estimate the effective surface area of the electrodes after each modification step and demonstrated in Table 2. where I p is the peak current, n is the number of electrons involved in the redox process, A is the surface area of the working electrode, D is the diffusion coefficient of the electroactive species, C * is the bulk concentration of the electroactive species and υ is the scan rate. As can be seen from obtained results in Fig. 4, the modified electrode exhibits an increase in effective surface area due to the increase in the slope of the anodic peak current vs ν 1/2 in different stages of surface modification 17 .
Electrodeposition of Au NPs. Figure 5a illustrates the electrodeposition of AuNPs on bare GCE by the CV technique. As shown in Fig. 5a, as the number of CV cycles increased, there was a decrease in the cathodic peak current, and the Epeak shifted toward lower negative potentials. This observation can be attributed to the dominant nucleation process of Au NPs on the GCE surface in the initial CV cycles. However, the growth of Au NPs becomes dominant in the subsequent CV cycles, making the electrodeposition easier 31 . The CV curves of the electro-reduction of Au(III) to precipitate Au(0) on MWCNTs/GCE in a 1 mmol L −1 HAuCl 4 solution is shown in Fig. 5b. As Fig. 5b shows the cathodic peak potential at MWCNTs/GCE (Epeak = 0.38 V) shifted to a lower negative potential compared to the bare GCE. This observation is attributed to the conductivity of MWC-NTs on the bare GCE, which facilitates electron transfer. Table 1. The EIS parameters extracted from the spectra of Fig. 3a were obtained in different modification steps of GCE. a The constant phase element as a circuit element was obtained from: Z CPE = 1/Q(j ω)g, where Q is the factor of proportionality, j is an imaginary number, g is CPE exponent (− 1 < g < 1), and ω = 2πf is the angular frequency. In the case g = − 1, CPE represents a pure inductor; g = 0, CPE is equivalent to a pure resistor; g = 0.5, CPE denotes diffusion behavior, and g = 1 indicates CPE is equivalent to a pure capacitor. b Double layer capacitance. www.nature.com/scientificreports/  The thickness of the cover layer was determined based on the concentration of MWCNTs, which was effective for the sensitivity of the detection method. Loading a specific amount of conductive MWCNTs enhanced the conductivity and chemical sensitivity of the electrode. Figure 6a shows that ∆R ct augments by increasing the concentration of MWCNTs and overtakes a maximum in the attendance of 0.02 mg mL −1 of MWCNTs dispersal due to the increase in the active surface area of the electrode and then decreases. The decrease in ΔR ct by increasing MWCNTs can be attributed to increasing the CNT-CNT contact resistance owing to the increasing thickness of the coated film. Thus, future analysis was performed in 0.02 mg mL −1 MWCNTs as the optimum concentration 32 .
Indirect determination of mesna based on a self-assembly interaction-free thiol group of Mesna with Au NPs on the modified electrode 33 . As shown in Fig. 6b, the ΔR ct values increased as the number of cycles increased from 2 to 15, due to an increase in active sites for strong bonds between the SH group of Mesna and Au NPs on the modified electrode. The ΔR ct values reached a maximum at 15 cycles, after which they began to decrease. This decrease may be attributed to the nucleation process stopping and the growth process starting for the Au NPs, leading to a decrease in active sites on the electrode surface 31 . Therefore, the 15 number of CV cycles for the reduction of Au(III) to Au(0) on MWCNTs/GCE was selected.
Optimization of pH. The solution pH can affect both the analyte structure and the electrode surface. Therefore, the optimization of the supporting electrolyte pH is essential before studying the electrochemical behavior of Mesna. Direct electrochemical detection of Mesna has not been reported in the literature. Therefore Optimization of incubation time. The incubation time for binding Mesna (30.0 nmol L −1 ) to the electrode surface was studied over a range of 0.5-5 min. The duration of incubation is a critical parameter that affects the interaction of Mesna with the electrode surface and, consequently, the sensitivity of the detection method. As shown in Fig. 8a,b, the ΔR ct value increased by increasing the time up to 2 min and then plateaued with a further increase in the incubation time 37 . Therefore, an incubation time of 2 min was chosen as the optimum time.  www.nature.com/scientificreports/ Analytical performance. The optimized conditions were used to evaluate the analytical performance of the proposed impedimetric sensor for various concentrations of Mesna. As shown in Fig. 9a, the EIS responses demonstrated an increase in the R ct with increasing concentrations of Mesna. Two linear dynamic ranges (LDRs) for Mesna determination were obtained (Fig. 9b). The first linear dynamic range, ranging from 0.06 to 1.0 nmol L −1 , has a slope of 486 Ω/nmol L −1 and an intercept of 731.51 Ω. The second linear dynamic range, ranging from 1.0 nmol L −1 to 130.0 µmol L −1 , has a slope of 143.39 Ω/nmol L −1 and an intercept of 715.95 Ω. The correlation coefficients (R 2 ) for the first and second LDR were 0.9974 and 0.9953, respectively. The first LDR is more sensitive, while the second LDR has a wider linear range. The self-assembly between Mesna and the electrode surface dominates at the first LDR and results in higher sensitivity. In other words, the number of active sites on the electrode surface is greater at lower concentrations of Mesna compared to higher concentrations of Mesna. Therefore, sensitivity decreases in the second LDR. Table 3 represents more detailed data on calibration curve analysis. The detection limit (DL) is defined as the lowest concentration of the analyte that can be reliably detected by the analytical method based on the blank signal. In the present study, the DL was determined as 3 times the standard deviation of the blank sample divided by the slope of the calibration curve, which was found to be 0.02 nmol L −1 . The obtained DL was lower than the acceptable concentration of Mesna in biological samples (i.e. 100 µmol  www.nature.com/scientificreports/ L −1 ) 38 , indicating the high sensitivity of the proposed sensor for the detection of Mesna in real samples. Also, a comparison between the proposed sensor and other methods 3,5-11 for the determination of Mesna is provided in Table 4. The linear range and limit of detection of the Au NPs/MWCNTs/GCE are better than those reported in previous methods. The presented impedimetric method has several notable features, including the synergistic effect of the electrocatalytic properties of Au NPs and MWCNTs, as well as the strong interaction between Mesna and the electrode surface, which enhances the sensitivity of the modified electrode.
Reproducibility and repeatability. The reproducibility of the Au NPs/MWCNTs/GCE was investigated using five parallel modified electrodes in a solution containing 1.0 nmol L −1 Mesna. The RSD% for the five parallel modified electrodes was found to be 4.6%. Since the thiol group of the drug interacts very strongly with the Au NPs, this results in the memory effect remaining on the electrode surface, which has both advantage and disadvantage. In the advantage, the Mesna drug interacts strongly with the electrode surface; in the disadvantage, the electrode surface must be polished and modified after each determination. Fortunately, the electrode surface can be modified in a simple and repeatable fashion, and it isn't costly or time-consuming. To check the repeatability of the proposed electrode surface modification, 10 glassy carbon electrodes were modified with AuNPs and MWCNTs and the Nyquist plots of [Fe(CN) 6 ] 3−/4− recorded at Au NPs/MWCNTs/GCE. According to Fig. 10, the RSD% for charge transfer resistance of [Fe(CN) 6 ] 3−/4− at Au NPs/MWCNTs/GCE was found to be 2.7%. It is clear from these results that the fabrication of the Au NPs/MWCNTs/GCE has proper repeatability. Furthermore, the analytical signal is the ∆R ct value , which is the difference between the charge transfer resistance from binding Mesna to modified electrode and the charge transfer resistance of Au NPs/MWCNTs/GCE as a blank signal. Due to this, there will be no problem in repeatability of the response in the measurements of Mesna.

Evaluation of interferences.
To evaluate the selectivity of the modified electrode, the ability of the proposed sensor to detect Mesna in the presence of some compounds commonly found in biological fluids was investigated ( Table 5). The limit of tolerance was determined as the highest concentration of interfering substances that caused an error of less than 5% in the measurement of Mesna   Table 6. The recoveries were in the range of 94-103.2% which is satisfactory. The results of the analysis of patient volunteers are shown in Table 7. The proposed Au NPs/MWCNTs/GCE was able to detect Mesna in the real samples (urine and serum) without any pre-concentration step. Additionally, different concentrations of Mesna were spiked into the real samples and satisfactory recoveries in the range of 93.5% to 106.6% were obtained. Additionally, to validate the performance of the method, the standard addition method was employed and the value of Mesna in the real sample of a cancer patient volunteer was obtained as 1.7 ± 0.09 µmol L −1 . These observations indicated that the proposed sensor can successfully assess the Mesna drug in the biological sample sans preparation. Therefore, Au NPs/MWCNTs/GCE can be suggested to determine Mesna in biological fluids.

Conclusion
This study presents a method to create an impedimetric sensor with excellent performance based on Au NPs and MWCNTs for the indirect detection of Mesna. To our best knowledge, there is no electrochemical determination of Mesna was reported still now. The fabrication of the modified electrode was confirmed with FESEM, EDX, and EIS methods, and the electrical conductivity and electrocatalytic ability of Au NPs and MWCNTs in the modified electrode allowed for high-sensitivity Mesna detection. The proposed method indirectly measured Mesna and achieved broad LDRs from 0.06 nmol L −1 to 1.0 nmol L −1 and 1.0 nmol L −1 to 130.0 µmol L −1 with a DL of 0.02 nmol L −1 . This impedimetric sensor showed excellent sensitivity, extremely low detection limit, and wide linear range in comparison with some reported methods. The suggested impedimetric sensor exhibits a lower DL than the levels of Mesna in real urine and serum samples, which suggests a potential for the expansion of impedimetric sensors for the indirect detection of other analytes. This could provide a simple and suitable method for the quantitative determination of the nanomole level of Mesna for clinical laboratories. Although this method is the first electrochemical method reported for the determination of Mesna drug in an indirect way, the used electrode cannot be regenerated after interacting with Mesna and the electrode surface should be modified for each measurement. Preparation of the real sample. Serum and urine samples were collected from cancer patients and healthy volunteers who were laboratory personnel. Before analysis with the proposed sensor, the biological samples were analyzed without spiking and then after spiking with known amounts of Mesna. To prepare the serum samples, 1 mL of acetonitrile was added to 1 mL of serum to precipitate serum protein, followed by centrifugation for 10 min at 4000 rpm. The supernatant was carefully collected and diluted with a B-R buffer solution to reach an optimum pH of 2.0 24 . Urine samples were also prepared by centrifuging at 4000 rpm for 15 min, followed by filtration with a Whatman 42 filter paper to obtain a clear solution, which was then diluted with a B-R buffer solution at the optimum pH of 2.0 18 .

Ethics approval. The study was approved by the ethics committee of the Hamedan University of Medical
Sciences (Bessat Hospital) in Hamedan, Iran. All subjects gave written informed consent and all methods were carried out following the Declaration of Helsinki.